In this experiment, all sheep were grown to sexual maturity on the farm and transported to the local slaughterhouse for slaughter (Jinzhong, Shanxi, China). Meanwhile, we collected the ovaries on-site and preserved them in DPBS, sterilized at 4 °C with 100 IU/mL of penicillin and 100 mg/mL of streptomycin (Solarbio, Beijing, China), and brought them back to the laboratory. The ovaries were sterilized with 75% alcohol, and follicles with a diameter of 3–5 mm were selected to remove the GCs for in vitro culture. Specific culturing methods were carried out as previously described (18). MNQ was isolated from the stems and leaves of Impatiens balsamina L., and its derivative D19 was synthesized based on the molecular structure of MNQ following previously reported methods (19). The structure of D19 was characterized by ¹H nuclear magnetic resonance (¹H NMR, 400 MHz, DMSO-d6), with characteristic peaks observed at δ 8.53 (s, 1H), 7.97–7.89 (m, 1H), 7.82 (d, J = 7.6 Hz, 1H), 7.73–7.68 (m, 1H), 7.57 (t, J = 7.5 Hz, 1H), 6.71 (dd, J₁ = 14.7 Hz, J₂ = 3.3 Hz, 1H), 6.58–6.44 (m, 2H), 4.28 (s, 1H), 4.20–4.00 (m, 3H), and 3.95 (s, 1H).

GCs were evenly suspended in a complete medium [89% DMEM-F12 (BOSTER, Wuhan, China), 10% fetal bovine serum (Cellmax, Beijing, China), and 1% penicillin–streptomycin (BOSTER, Wuhan, China)], inoculated in cell culture plates of different sizes and cultured in a 37 °C and 5% CO₂ humidified incubator. The inoculum densities of GCs in 96-well and 6-well plates were 1 × 10⁴ cells/well and 1 × 10⁶ cells/well, respectively (20). GCs were treated with D19 (prepared as a 10 mM stock solution in DMSO; final DMSO concentration <0.1%) at concentrations of 0, 0.01, 0.1, 1, 10, 100, and 200 μM. Control groups (CK) received equivalent DMSO concentrations without D19. All groups were incubated for 24 h and 48 h, respectively (17, 21). GCs were incubated with different concentrations of LPS (0, 10, 25, 50, 100, 200, 400 μg/mL) (Solarbio, Beijing, China) for 12 h and 24 h (22). Control siRNA (si-NC: sense, 5′-UUCUCCGAACGUGUCACGUTT-3′; antisense, 5′-ACGUGACACGUUCGGAGAATT-3′) and GPX4-targeting siRNA (si-GPX4: sense, 5′-AAGAGUUCGCUGCUGGCUA-3′; antisense, 5′-UAGCCAGCAGCGAACUCUU-3′) were synthesized by Shanghai Sangon Biotech. GCs were transfected with 20 μM of either si-NC or si-GPX4 using an RNA transfection reagent (Sangon, Shanghai, China). Transfection was performed for 12, 24, 36, or 48 h. The experimental groups included: (1) CK, (2) LPS (cells treated with LPS for 12 h), (3) LPS + D19 (cells treated with LPS for 12 h, followed by D19 treatment for an additional 24 h), (4) si-NC (cells transfected with control siRNA for 36 h), (5) si-GPX4 (cells transfected with GPX4-targeting siRNA for 36 h), and (6) si-GPX4 + D19 (0.1 μM) (cells transfected with GPX4-targeting siRNA for 12 h, followed by D19 treatment for an additional 24 h).

The immunofluorescence experiments were performed as previously described (23). Anti-FSHR (Bioworld, Minnesota, United States) and anti-GPX4 (Sangon Biotech, Shanghai, China) were used as primary antibodies, and goat anti-rabbit IgG (BOSTER, Wuhan, China) was used as the secondary antibody. The negative control group was incubated with PBS instead of the primary antibody. Specifically, GCs were inoculated into six-well plates, and the medium was removed and replaced with 4% paraformaldehyde fixation when the cells had fused to 60–70%, and GCs were permeabilized with 1% TritonX-100 (Solarbio, Beijing, China) after washing with PBS, followed by sealing with 1% BSA (Solarbio, Beijing, China). GCs were incubated with anti-FSHR and anti-GPX4 antibodies overnight at 4 °C. Hoechst 33342 was used to stain the nuclei of the cells, and the antigen–antibody conjugate reaction was performed with anti-rabbit IgG, and then placed under a fluorescence microscope to observe the specific protein expression.

The toxic effects of different concentrations of D19 and LPS, as well as si-GPX4 on the cells at different time intervals, were performed in strict accordance with the instructions of the MTT kit (Solarbio, Beijing, China). GCs viability of each experimental group was calculated using the OD detected at 490 nm.

The senescence of GCs in each treatment group was detected according to the instructions of the SA-β-galactosidase (SA-β-Gal) staining kit (Beyotime, Shanghai, China). Specifically, GCs were removed from the culture and added to β-galactosidase staining fixative, fixed at room temperature for 15 min, washed with PBS, and then the prepared working solution (1% β-galactosidase staining solution A, 1% β-galactosidase staining solution B, 93% β-galactosidase staining solution C, and 5% X-Gal solution) was incubated overnight at 37 °C. The senescent cells were visualized under a light microscope to produce the Dark blue color results.

Glucose content and LDH activity in treated GCs were assayed in strict accordance with the Glucose kit (Njjcbio, Nanjing, China) and Lactate dehydrogenase assay kit (Njjcbio, Nanjing, China).

The experimental procedure was performed as previously described (24). Briefly, the treated GCs were pre-fixed with 3% glutaraldehyde for about 24 h and 1% osmium tetroxide for another 2 h; dehydrated and osmotically embedded according to the concentration gradient, and then stained after ultrathin sectioning using an ultrathin sectioning machine, and finally the images were captured and analyzed in the electron microscope.

Cultured GCs (treated) complete medium was replaced with 2 μM JC-1 (MCE, Shanghai, China) and continued to be cultured in the medium for 15–20 min, washed with PBS, and then the staining results were observed by fluorescence microscopy (green fluorescence: Ex/Em = 510/527 nm; red fluorescence: Ex/Em = 585/590 nm).

The levels of steroid hormones (A₄, E₂, and P₄) and inflammatory factors (TNF-β, IL-1β) in the treated GCs were measured according to the instructions of the ELISA kit (MEIMIAN, Jiangsu, China). Specific experimental steps were performed as previously described (25). Briefly, the diluted GCs supernatant was labeled with horseradish peroxidase (HRP), incubated for 1 h at a constant temperature of 37 °C, washed five times with washing solution, and then reacted with substrates A and B simultaneously for 15 min. The reaction was terminated by adding a termination solution. Then, the OD value at 450 nm was measured in each well, and the final concentration was calculated according to the standard curve. It is worth noting that the procedure was the same for all five assays, all concentrations were normalized to 2.5 × 10⁶ cells, and all experiments were repeated at least three times.

The antioxidant capacity of GCs in different treatment groups was detected by using Nanjing Jianjian Bioengineering Institute kits (including SOD, GSH, GSH-PX, MDA, total antioxidant capacity, CAT, and ROS) and Lipid Peroxidation Assay Kit (BODIPY 581/591 C11) (Beyotime, Shanghai, China) and operated according to the specifications. All experiments were repeated at least three times.

A ferrous ion content assay kit (Solarbio, Beijing, China) was used to measure Fe²⁺ levels in GCs after adding different treatments. The OD value at 593 nm was substituted into the standard curve to calculate the final results, and each set of experiments was repeated at least three times.

Total RNA was extracted from GCs using the Mei5bio RNA extraction kit (Mei5bio, Beijing, China), and cDNA synthesis was performed according to the protocol provided with the PrimeScript RT Reagent Kit (Takara, Tokyo, Japan). Quantitative real-time PCR (qRT-PCR) was conducted on a Bio-Rad CFX instrument using the TransGen PerfectStart^® Green qPCR SuperMix kit (TransGen, Beijing, China), following the manufacturer’s instructions. Primer sequences were designed and synthesized by Shanghai Sangon Biotech (see Table 1 for details). The qRT-PCR protocol included an initial denaturation step at 95 °C for 60 s, followed by 40 cycles of amplification (95 °C for 30 s, 95 °C for 5 s, and annealing at the primer-specific Tm for 30 s) (25). β-actin was used as an internal control, and each experiment was performed in triplicate to ensure reproducibility. Relative gene expression levels were calculated using the 2^−ΔΔCt method.

Total GC proteins were extracted with ready-to-use lysate (98% RIPA, 1% PMSF, 1% broad-spectrum phosphorylated protease inhibitor), centrifuged, and added to the protein sample buffer at a ratio of 4:1. After SDS-PAGE electrophoresis (80 V for 40 min, 120 V for 90 min), the proteins were transferred to NC membranes (120 V, ice bath for 90 min). The membrane was blocked with skimmed milk powder for 1 h, the primary antibody was incubated overnight at 4 °C, and the secondary antibody (LI-COR, Lincoln, NE, United States) was incubated at room temperature and protected from light for 1 h. The above reagents were purchased from BioTech. Protein bands were imaged using the Odyssey Infrared Imaging System and ImageJ to analyze relative changes. β-actin was used as an internal reference protein. Antibody information is shown in Table 2.

Data were analyzed statistically using SPSS 22.0, subject to normality and chi-square: one-way ANOVA with Tukey’s-b or Dunnett’s post-hoc multiple comparisons between groups; t-tests for independent samples were used between groups. GraphPad Prism 8.0 was used for graphical presentation. Data are presented as mean ± SEM. p < 0.05 was the threshold for statistical significance, and all experiments were repeated at least three times.

Immunofluorescence staining showed that the FSHR protein displayed particular expression in sheep follicle GCs, demonstrating that the GCs used in this study possess high purity and activity and are appropriate for subsequent experiments (Figure 2A).

Figure 2B demonstrates a concentration-dependent effect of LPS on GC viability, with the optimal survival rate observed at 25 μg/mL (p < 0.05). Then, the survival rate of the cells decreased with the increase in the concentration. When the concentration of LPS reached 400 μg/mL, the GCs were stressed, and some cells died. The results of GCs treated with different concentrations of D19 showed that low concentrations of D19 (0–0.1 μM) accelerated the activity of GCs (Figure 2C). The survival rate of GCs was significantly increased (p < 0.05) after treatment with 0.1 μM D19 for 24 h and 48 h. On the contrary, high concentrations (1–200 μM) of D19 caused dose-dependent cytotoxicity. Approximately 80% of GCs at 200 μM showed morphological abnormalities and were detached (p < 0.05). Since 400 μg/mL LPS would damage the activity of GCs, the present study was carried out to investigate the optimal therapeutic concentration and safety range of D19 at different concentrations. Our findings indicate that 0.1 μM D19 is the optimal therapeutic concentration, effectively mitigating LPS-induced damage in GCs (p < 0.05). However, D19 exhibited a narrow therapeutic window, with concentrations exceeding 100 μM failing to provide any protective benefit and instead demonstrating significant toxicity (Figure 2D).

To explore the mitigating effect of D19 on LPS-induced inflammation in GCs, the mRNA expression levels of IL-6, IL-1β, and TNF-α in GCs treated with different concentrations of LPS were first detected. The results showed that LPS dose-dependently increased the mRNA expression of the above inflammatory factors compared with CK (p < 0.05), with the maximum observed at a concentration of 400 μg/mL (Figure 3A). In subsequent experiments, 400 μg/mL LPS was used to induce inflammation in GCs. Further studies revealed that D19 was able to inhibit the LPS-induced inflammatory response of GCs. Compared with the LPS group, 0.1 μM D19 significantly reduced the mRNA levels of IL-6, IL-1β, and TNF-α (p < 0.05), and the therapeutic effect at this concentration was better than that of other concentrations (Figures 2D, 3B). In addition, 100 μM of D19 was the maximum safe concentration for treating inflammation in combination with cell activity assay (Figures 2D, 3B). D19 was found to significantly inhibit the LPS-induced elevation of IL-1β and TNF-α levels in GCs by ELISA kit assay (p < 0.05) (Figure 3D), and the results of protein levels were consistent with the above findings (Figures 3B,C). Notably, SA-β-Gal staining revealed that the dark blue products in GCs were significantly reduced in the LPS + D19-0.1 μM and LPS + D19-100 μM groups compared to the LPS group, indicating that D19 alleviated LPS-induced cellular senescence (Figure 3E). Moreover, the effect of D19 was more pronounced at 0.1 μM than at 100 μM (Figure 3E). The above studies confirmed that D19 reversed LPS-induced inflammatory injury in sheep follicular GCs.

As shown in Figure 4A, 400 μg/mL LPS significantly inhibited the expression of antioxidant-related genes GPX4, CAT, and SOD2 in sheep follicular GCs (p < 0.05). D19-0.1 μM effectively upregulated the expression levels of the genes mentioned above, with its effective concentration extending up to 100 μM (p < 0.05) (Figure 4B). Western blotting analysis (Figure 4C) confirmed that 0.1 μM D19 could effectively alleviate the LPS-induced reduction in the protein expression of GPX4, CAT, GSH-PX, and SOD2 (p < 0.05). 100 μM D19 also had antioxidant effects, but the effect was weaker than that of 0.1 μM (p < 0.05). In addition, 400 μg/mL LPS effectively inhibited the total antioxidant capacity, SOD activity, GSH-PX activity, CAT activity, and GSH level of GCs, and significantly increased MDA content, glucose content, LDH level, and ROS production. D19 could significantly reverse the above LPS-induced weakening of antioxidant indexes in GCs (Figures 4D,E). These data demonstrate that D19 protects sheep follicular GCs from LPS-induced oxidative damage.

Given that GPX4, a core molecule in ferroptosis regulation, has been significantly inhibited by LPS in this study, and combining the above findings, we speculate that the protective effect of D19 against LPS-induced functional impairment of GCs may be realized through the GPX4-mediated ferroptosis signaling pathway. As depicted in Figure 5A, LPS treatment significantly decreased the mitochondrial membrane potential, as evidenced by enhanced green fluorescence, whereas the mitochondrial membrane potential in the LPS + D19-0.1 μM and LPS + D19-100 μM groups was close to the normal level. TEM results (Figure 5B) revealed that GCs in the LPS-treated group displayed significant ultrastructural abnormalities compared with the CK group. These abnormalities included mitochondrial condensation, reduced volume, reduction and coarsening of cristae, widening of the inter-cristae lumen, and increased membrane and electron densities. Furthermore, autophagic lysosomes and a small number of vacuoles were observed. Compared with the LPS group, LPS + D19-0.1 μM significantly improved mitochondrial morphology, with most of the mitochondria restored to an elliptical shape, uniform matrix electron density, and well-defined and straight cristae. The morphology of LPS + D19-100 μM cells was slightly abnormal, and the mitochondria showed a slight condensation, accompanied by a mild expansion of the rough endoplasmic reticulum. Autophagic lysosomes were still visible in the cytoplasm. In addition, the large accumulation of Fe²⁺ in LPS-treated cells was significantly reversed by D19 treatment (Figure 5C), which also successfully alleviated the inhibitory effect of LPS on FTH1 mRNA and protein expression levels (p < 0.05) (Figures 5D,E). Taken together, these results suggest that D19 can effectively alleviate the process of LPS-induced ferroptosis in sheep follicular GCs and mitigate cellular damage.

To assess the effect of D19 on LPS-induced steroid hormone synthesis in GCs, we examined the levels of A₄, E₂, and P₄ in GCs. The ELISA results showed that LPS significantly inhibited the synthesis of these three hormones in GCs compared to CK (p < 0.05), whereas D19 treatment effectively reversed the inhibitory effect of LPS and significantly elevated the A₄, E₂, and P₄ expression levels (p < 0.05) (Figure 6A). The expression of steroid synthesis-related genes (HSD17B4, CYP19A1, 3β-HSD, CYP11A1, and STAR) and their proteins were also greatly inhibited by LPS, and the expression levels of these genes and proteins were significantly increased by D19 treatment (p < 0.05) (Figures 6B,C). Based on these findings, we conclude that D19 effectively mitigates LPS-induced impairment of steroid hormone synthesis in sheep follicular GCs.

GPX4 protein is specifically expressed in sheep follicular GCs (Figure 7A). si-GPX4 effectively inhibited GPX4 mRNA and protein expression (p < 0.05) (Figures 7B,C) and significantly reduced cell viability at multiple time points (p < 0.05) (Figure 7D). However, 0.1 μM D19 effectively alleviated the inhibitory effect of si-GPX4 on GC activity (p < 0.05) (Figure 7D). To further study the effect of GPX4 deletion on the inflammatory response of GCs and to clarify the role of D19, we obtained the following findings: ELISA results (Figure 7E) revealed that D19 effectively reversed the si-GPX4-mediated upregulation of IL-1β and TNF-α levels in GCs (p < 0.05). Mechanistically, we found that si-GPX4 activated the NF-κB signaling pathway, indicated by a significant increase in NF-κB mRNA expression and p65 phosphorylation, subsequently promoting the expression of TNF-α, IL-6, and IL-1β (p < 0.05) (Figures 7F,G). Notably, total p65 protein levels remained unchanged across all groups (p > 0.05) (Figure 7G). The protein levels of TNF-α and IL-1β were consistent with those of mRNA (p < 0.05) (Figures 7F,G). Interestingly, after D19 treatment, the expression levels of NF-κB, TNF-α, IL-6, and IL-1β were significantly lower than those in the si-GPX4 group (p < 0.05) and returned to near-normal cell levels. Meanwhile, Western blotting results showed that the trends of protein levels of p65 NF-κB, TNF-α, and IL-1β were consistent with the mRNA results (Figure 7G). Therefore, we conclude that D19 can effectively alleviate si-GPX4-induced decrease in cell viability and inflammatory injury.

To investigate whether D19 protects against LPS-induced oxidative damage via GPX4, we suppressed GPX4 expression in sheep follicular GCs and assessed oxidative stress and D19’s effects. Compared with GCs transfected with si-NC, antioxidant indices (total antioxidant capacity, SOD activity, CAT activity, GSH-PX activity, and GSH level) were significantly decreased (p < 0.05), while oxidative damage and metabolism-related indices (ROS level, MDA content, glucose level, and LDH activity) were significantly increased (p < 0.05) in GCs with GPX4 knockdown (Figures 8A,B). Si-GPX4 significantly reduced mRNA and protein expression levels of antioxidant-related genes (NRF2, CAT, SOD2, and GPX4) compared to si-NC (p < 0.05), as shown in Figures 8C,D. After the D19 treatment, the above antioxidant indexes were significantly restored (Figure 8B), oxidative damage and metabolism-related indexes were reduced (Figures 8A,B), and the expression levels of antioxidant-related genes and proteins were effectively up-regulated (p < 0.05) (Figures 8C,D). These data demonstrate that D19 has therapeutic potential for treating si-GPX4-induced oxidative damage in GCs.

Evidence indicates that intracellular inflammatory responses and oxidative stress are strongly linked to ferroptosis caused by GPX4 downregulation. Thus, we investigated whether D19 alleviates GPX4 deficiency-induced inflammation and oxidative damage in GCs by modulating ferroptosis. Suppressing GPX4 dramatically elevated Fe²⁺ concentration in GCs compared to the si-NC group (p < 0.05), as demonstrated in Figure 9A. However, D19 treatment in si-GPX4-transfected GCs normalized Fe²⁺ levels, indicating D19’s ability to rescue GCs from GPX4 deficiency-induced iron overload (see Figure 9A). As shown in Figure 9B, si-GPX4-transfected GCs exhibited a significantly increased green fluorescence signal compared to the si-NC group, indicating elevated lipid peroxide levels. D19 treatment attenuated this increase, resulting in a red fluorescence signal distinct from the si-GPX4 group, suggesting that D19 effectively reduced lipid peroxide levels. TEM analysis showed that si-GPX4 GCs exhibited significant mitochondrial dysfunction, indicated by decreased membrane potential, cristae loss, increased membrane density, and elevated autophagosome numbers. D19 treatment mitigated these abnormalities, restoring mitochondrial morphology and membrane potential (Figures 9C,D). In addition, the gene and protein expression of FTH1 in the GCs of the si-GPX4 group was significantly lower than that of the si-NC group (p < 0.05), whereas the expression level of FTH1 in the si-GPX4 + D19 group was significantly higher than that of the si-GPX4 group (p < 0.05) (Figures 9E,F). Therefore, we conclude that D19 can effectively inhibit the ferroptosis process induced by GPX4 deficiency and maintain the iron metabolism homeostasis in GCs.

The critical role of GCs in follicular development is realized through the synthesis of steroid hormones. However, when GCs were exposed to an environment in which GPX4 was inhibited, the synthesis of E₂ and P₄ was significantly suppressed compared with the si-NC group (p < 0.05) (Figure 10A), while the mRNA and protein expression levels of steroid hormone synthesis-associated factors (HSD17B4, CYP19A1, 3β-HSD, CYP11A1, and STAR) were also significantly downregulated (p < 0.05) (Figures 10B,C). Noticeably, the levels of E₂ and P₄, as well as the expression of steroid hormone synthesis-related genes and proteins, were significantly up-regulated in the si-GPX4 + D19 group compared with the si-GPX4 group (p < 0.05) (Figures 10A–C). These results suggest that D19 can effectively alleviate the inhibition of steroid hormone synthesis function in GCs by GPX4 deletion and thus repair the functional damage of GCs.